Two classes of proteins mediate glucose uptake in mammalian cells: the glucose transporters and the hexokinases. Transport is often thought to be rate-limiting, but the phosphorylation of glucose may play a larger role in the regulation of glucose metabolism than has previously been considered. There are four major hexokinases (HKI-IV) in mammalian cells. The focus of this proposal is HKII which, like HKI and HKIII, is a 100kD isoform that is inhibited by physiologic concentrations of G-6-P. HKII is the most abundant isoform found in adipose tissue, skeletal muscle and heart; these are tissues in which insulin stimulates glucose uptake and utilization. Earlier studies showed that insulin increases HKII activity in these tissues, but nothing was known about the structure or regulation of the gene. In the past project period we isolated and characterized the HKII cDNA and gene and showed that insulin and cAMP increase HKII gene transcription in muscle and adipose cells. We also learned something about how the HK family of proteins evolved, and showed that HKII is quite different in its kinetic characteristics from the other 100 kDa HKs. This proposal includes studies designed to continue a comprehensive approach toward understanding how the hexokinase II gene (HKII) is regulated, how the structure of the protein relates to its function, and how HKII contributes to glucose metabolism. Two Specific Aims are presented. Aim 1 concerns the regulation of the HKII gene by insulin. Several experiments are proposed to define how this hormone acts through complex, multicomponent hormone response units, and the several transcription factors that appear to be involved. We also propose studies aimed at defining the signal transduction pathway from the insulin receptor to the HKII gene. HKII is unique among the 100 kDa hexokinases in that it actually consists of two tandemly linked enzymes, each with different kinetic properties. These two halves of the molecule show cooperative behavior toward the product inhibitor G-6-P. A goal of Aim 2 is to define the structural features required for this effect and to analyze how this kinetic feature may influence the activity of the enzyme in intact cells. A novel, cell-based reporter assay has been developed for assessing the function of various mutated and chimeric hexokinases in intact cells. We plan to use laser-based two photon microscopy to analyze whether insulin promotes the association of HKII with mitochondria, and whether this affects glucose metabolism [measured as NADC(P)H fluorescence] in single cells. We recently showed that HKII gene regulation is abnormal in non-insulin dependent diabetes. The studies in this proposal may help us understand how impaired peripheral glucose utilization, primarily in skeletal muscle, contributes to the pathophysiology of this disease.